1 / 34

Swetha Charles Jessy Labb é

Phenotyping the effect of mutant P. fluorescens on mycorrhizal development and function of L. bicolor with Populus. Swetha Charles Jessy Labb é. What’s coming?. Which P. fluorescens genes are involved inthe helper effect?. Plant Microbe Interface (PMI).

giulio
Download Presentation

Swetha Charles Jessy Labb é

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Phenotyping the effect of mutant P. fluorescens on mycorrhizal development and function of L. bicolor with Populus Swetha Charles Jessy Labbé

  2. What’s coming? • Which P. fluorescens genes are involved inthe helper effect?

  3. Plant Microbe Interface (PMI) The PMI group is studying the relationship that exists between plants and their microbial environment. One key area of study is to understand the genetic foundation that underpins molecular communication between Populus trees and their microbial system.

  4. Why Populus? It is the first tree genome that was sequenced. Many tools are available for genetic manipulation. It’s one of the prime candidates for bioenergy production and for studying carbon sequestration. It is found in many places in the USA

  5. Why Populus? A good understanding of microbial interactions with Populus will be useful across many areas.

  6. What is mycorrhizal symbiosis? A simple explanation: An interaction between plants and fungi where both partners benefit [Barbieri] Cusano, pg 204

  7. Mycorrhizal Symbiosis • Mycorrhizas, rather than roots, are responsible for nutrient uptake in land based plants [Smith] • Mycorrhizal symbiosis is the normal state for most plants

  8. Mycorrhizal Symbiosis • Except in a few cases, the fungi obtains most of its organic C from the plant [Smith] • With this advantage, the mycorrhiza can compete with other organisms in the soil for nitrogen, phosphate and other nutrients • ECM fungi prefer sources of inorganic N • This increases plant access to nutrients and allows growth in suboptimal conditions

  9. Mycorrhizal Symbiosis There are three main types of Mycorrhizal symbiosis [Smith] : • Arbuscular mycorrhiza (endomyccorhiza) • Ectomycorrhiza • Ericoid, orchid and mycoheterotrophic mycorrhiza(some are endomycorrhiza) Populus trees form relationships with both endo- and ectomycorrhizas. • The latter relationship is our focus.

  10. Mycorrhizal helper bacteria Mycorrhizal fungi are surrounded by microbial communities which affect mycorrhizal symbiosis. [Deveau] Garbaye, 1994

  11. Mycorrhizal helper bacteria The creation of the mycorrhizal relationship is substantially improved in the presence of certain soil bacteria The bacteria also help support the continuing symbiosis Garbaye, 1994

  12. How do MHB help mycorrhizal formation? • Increase root receptivity for infection by fungi • Improve the recognition process between roots and fungi • Stimulate fungal growth before symbiosis occurs • Modify the rhizosphere making it more conducive for mycorrhizal infection • Induce fungal spore germination[Deveau] Garbaye, 1994

  13. Pseudomonas fluorescens characterisation The P. fluorescens strain BBc6R8 has been used to study mycorrhizal formation in Douglas fir trees It was initially recovered from a sporocarp of Laccaria bicolor A test of the microbial communities in Populus soil showed the presence of Laccaria bicolor

  14. Pseudomonas fluorescens characterisation • After transcriptome and genome analysis, two sets of gene clusters in P. fluorescens (BBc6R8) were found to be necessary for optimum mycorrhizal development with Laccaria bicolor • T3SS system • Co-regulated flagellar system

  15. Type three secretion system (T3SS) Pseudomonas fluorescens characterisation The T3SS system was checked by Cusano et al and proved important for MHB effect. Cusano, pg 204

  16. Co-regulated flagellar system For the flagellar system, the cluster underwent targeted mutagenesis to create different mutant strains. • Mutants are created by tn5 insertions • elements known as transposons are inserted into the gene cluster, disabling certain genes http://www.cambio.co.uk/216/10/products/ez-tn5-andlt;t7/kan-2andgt;-promoter-insertion-kit/

  17. Populus, Laccaria bicolor and Pseudomonas fluorescens (BBc6R8) • My project will be in parts and will focus on a model system: • Project 1: co-cultivation of L. bicolor and P. fluorescens • Project 2: co-cultivation of L. bicolor and P. fluorescens mutants with Populus species

  18. Project 1: Laccaria Bicolor and P. fluorescens Fungal plug in the centre and bacteria placed in four spots equidistant from the plug

  19. Characteristics to measure Fungal diameter of Laccaria bicolor with no bacteria Day 4 Day 8 Day 11 Day 14 Day 18 Day 22 Day 25

  20. Characteristics to measure Fungal diameter of Laccaria bicolor with mtP464 Day 4 Day 8 Day 11 Day 14 Day 18 Day 22 Day 25

  21. Fungal diameter of Laccaria bicolor with mtP464 vs. mt4delta1 mt4delta1 (Day 25) mtP464 (Day 25)

  22. Higher than wild-type growth rate: bacteria as food source in nutrient limited environment Receptor Signal Response

  23. L. bicolor with no bacteria Receptor Signal Response

  24. L. bicolor with wild type bacteria Receptor Signal Response

  25. L. Bicolor withmutant bacteria Receptor Signal Response

  26. Branching density BBc6R8 (Day 25) L. bicolor (Day 25)

  27. Branching density mtP464 (Day 25) mt4delta1 (Day 25)

  28. Expected Results • We hope shed more light on the P. fluorescens genes that are important for myccorhizal development and function: • The mutants which have no helper effect maypoint to genes of interest • Mutants which have a higher than wt growth rate may point to the fungus gaining nutrients by eating the bacteria • Previous proof of concept project highlighted mtP464 as a possible focus

  29. Project 2: Testing a new phenotype by inoculating three species of poplar with L. Bicolor and 3 promising mutants Populus species: • P. trichocarpa • P. deltoides • P. tremuloides(P. tremula X P. alba) P. fluorescensmutants: • mtP464 • mt4delta1 • mt4delta2

  30. Other contributions • BESC – Inoculating promising P. fluorescensmutants with P. deltoides mutants • The Populus mutants are aimed at reducing recalcitrance to increase the ease ethanol production • Bioinformatics: • Look for known transposable elements in the genome sequence of 17 fungal isolates: • Check for polymorphisms in the transposable elements across the isolates • Highlight areas where the transposable elements and symbiosis genes overlap or occur in close proximity • Search for polymorphisms in symbiosis genes across the 17 isolates • Methods paper: • Summary of techniques used for phenotyping experiments

  31. References Deveau, A., Palin B., et al. The myccorhiza helper Pseudomonas fluorescensBBc6R8 has a specific priming effect on the growth, morphology and gene expression of the ectromycorrhizal fungus Laccaria bicolor S23N. New Phytologist, 2007. 175, 732-755. Barbieri, E., Ceccaroli, P., et al. Ectomycorrhiza associated bacteria: the third partner in the symbiosis. Cusano, A.M., Burlinson, P., et al. Pseudomonas fluorescens BBc6R8 type III secretion mutants no longer promote ectomycorrhizal symbiosis.  Environmental Microbiology Reports, 2010. 3:203–210 Smith, S., Read, D. Myccorhizal Symbiosis: third edition. 2008. Garbaye J.  Helper bacteria: a new dimension to the mycorrhizal symbiosis. New Phytologist, 1994. 128: 197–210

  32. Acknowledgements JessyLabbé Lee Gunter Zack Moore Julie Malicoat Cheryl Brown Tim Sullivan

  33. Questions?

More Related